1. Field of Invention
The present invention provides a method that reduces or eliminates nonspecific primer extension products. More specifically, the method uses single-stranded nucleic acid binding proteins to reduce or eliminate these products. This invention is contemplated to be especially useful as a novel Hot Start method for the polymerase chain reaction (PCR).
2. Description of Related Art
Amplification of nucleic acids is of fundamental importance in modern science. During this process, nucleic acids are duplicated or replicated through coordinated, catalytic synthesis.
In general, nucleic acid amplification occurs through a process of hybridizing (annealing or pairing) a relatively short single-stranded nucleic acid (primer or oligonucleotide), to a relatively longer single-stranded nucleic acid counterpart (target or template) that has complementary nucleic acid sequence. Complementary annealing refers to the base pairs which form and are stabilized by hydrogen bonds described by Watson-Crick pairing rules (i.e., A-T and G-C base pairs). A polymerase can use this hybrid (or complement) to catalytically add bases or nucleotides which are present in the reaction to the 3′ end of the primer. The nucleotides are added such that they are complementary to the target or template. Since the newly synthesized strand of nucleic acid is the result of nucleotides which extend the length of the primer, this process is also known as primer extension. To be extended by a polymerase, a primer strand first must be annealed to a template strand.
Although the primer(s) used in primer extension reactions are designed to be complementary to a specific portion of the template strand, under certain conditions the primer can and will anneal to other regions of the template strand with which it is only partially complementary, or in rare cases, noncomplementary. As used herein, a fully complementary pairing is referred to as and is the result of specific priming and a partially complementary (or noncomplementary) pairing is referred to as and is the result of nonspecific priming. Since the polymerase cannot discriminate between partial versus full complements, primer extension products can and will be formed from both if both are present under extension conditions. As used herein, primer extension products from full complements are referred to as specific products and those from partial (or non-) complements are referred to as nonspecific products.
The degree to which a primer will hybridize to full versus partial (or non-) complementary sequences is governed by well-known principles of thermodynamics. A useful parameter is known as the melting temperature (Tm) and is defined as the temperature at which 50% of the primer and its true complement or intended target sequence is annealed. The most common method to determine the actual Tm is to plot temperature versus absorbance in a UV spectrophotometer (e.g., Marmur and Doty, 1962, Journal of Molecular Biology 5:109-118). This empirical determination is often not practical and thus theoretical methods have been devised to predict melting temperatures. One such method is through an equation known as the Wallace Rule (Suggs et al., 1981, In Developmental Biology using Purified Genes 23:683-693). This equation states that Tm (in ° C.) is approximately equal to 2×(#A+#T)+4×(#G+#C), where # is the number of A, G, C, or T bases present in the primer. Thus, a primer 20 bases long with an equal base content would be predicted to have a Tm of 2×(5+5)+4×(5+5)=60° C.
Although other factors such as salt concentration, DNA concentration, and the presence of denaturants affect the melting temperature, the main contribution to Tm is from the length and base composition of the primer. Given a defined primer sequence, the temperature of the hybridization reaction determines the amount of specific versus nonspecific priming based on thermodynamic principles. Temperatures significantly below the Tm will permit nonspecific priming while temperatures significantly above the Tm will restrict nonspecific and specific priming (e.g., Gillam et al., 1975, Nucleic Acids Research 2(5):625-634; Wallace et al., 1979, Nucleic Acids Research 6(11):3543-3557). Ideally, hybridization is carried out at or near the Tm of the primer(s) to generate specific complements and thus specific primer extension products. As used herein, hybridization and primer extension temperatures significantly lower than the Tm of the primers are referred to as permissive or nonstringent while temperatures at or near the Tm are referred to as restrictive or stringent. Thus, permissive or nonstringent temperatures lead to nonspecific primer extension products while restrictive or stringent temperatures lead to specific ones.
A well-known example of primer extension is the polymerase chain reaction (PCR). In this technique, DNA synthesis occurs in a series of steps comprising a cycle, this cycle being repeated many times to amplify the primer extension reaction products for further analyses. Two primers typically are used in which their respective 3′-ends face one another to generate a double-stranded DNA product whose length is defined as the distance between the primers. Typically, the cycle consists of a step which generates single-stranded DNA, a step which allows primers to hybridize with their target sequences, and a subsequent step for primer extension by the polymerase. The PCR technique is described in detail in U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,965,188. A variant of PCR, which is called reverse transcription-PCR (RT-PCR), is when RNA is used as a template in the reaction instead of DNA. In this technique, an initial step of converting the RNA template to DNA is performed with a polymerase which has reverse transcriptase activity. Following this initial template conversion (reverse transcription step), reactions proceed as in standard PCR.
Each cycle of PCR generates a geometric expansion of the original target (i.e., doubling per cycle), which after the 25-50 cycles typically employed in PCR can amplify the target well over a billion times. Unfortunately, amplification from nonspecific priming can also occur which is detrimental since these nonspecific products may obscure specific ones. The specificity of the PCR depends on many factors, but as previously discussed, the temperature of the hybridization and subsequent extension steps is important in obtaining specific primer extension products. Fortunately, the discovery and widespread use of thermostable polymerases, such as the polymerase from Thermus aquaticus (Taq DNA Polymerase), allows the use of more stringent reaction temperatures (Chien et al., 1976, Journal of Bacteriology 127(3):1550-1557; Saiki et al., 1988, Science 239(4839):487-491). Stringent hybridization temperatures increase the probability of generating specific products.
Although the temperatures used during the polymerase chain reaction can be stringent, the reaction mixtures themselves are not conveniently assembled at higher temperatures, temperatures at which greater priming specificity occurs. PCR reactions are usually assembled at lower temperatures such as on ice or most preferably at room temperature (i.e., 20-25° C.). If the average primer can be assumed to have a Tm of about 50-60° C., the temperatures at which reaction set-up occur are clearly significantly lower and will favor nonspecific priming. At room temperature, the conventional polymerases used in the PCR (e.g., Taq DNA Polymerase) have some degree of catalytic activity which leads to the synthesis of nonspecific reaction products. In addition, even if the reactions are assembled on ice, they must be placed in a machine which provides the temperatures necessary for cycling. Stringent hybridization temperatures higher than ice cannot be achieved instantaneously and nonspecific products can also be generated during this “ramping” stage. At permissive temperatures primers not only pair nonspecifically with the template but also pair with other primers leading to nonspecific primer extension products known as “primer-dimers.” Nonspecific amplification is a ubiquitous problem during the assembly of polymerase chain reactions and is covered in greater detail in Chou et al., 1992, Nucleic Acids Research 20(7):1717-1723.
Since nonspecific amplification products can be generated during assembly of PCR reactions, a method is needed that can reduce or eliminate these artifacts. Various methods have been developed to address this problem. These techniques are generally known as “hot-start” methods because the primer extension reactions are not allowed to “start” until stringent or “hot” hybridization temperatures have been reached. Several of these methods are briefly described below.
In the simplest hot-start method, one of the critical components for successful DNA synthesis is omitted from the reaction mixture during preparation at room temperature. Then, the omitted component is added manually, as through pipetting, after the temperature of the reaction mixture has reached, or more usually exceeded, a threshold stringent temperature based upon the Tm of the primer(s). This method is often called manual hot-start PCR. For example, one may omit the polymerase or the divalent cation (e.g., Mg2+) which is essential for polymerase activity from the reaction mixture until the stringent temperature is reached or exceeded. Because a key component is unavailable at lower temperatures, nonspecific extension products cannot be formed. This method is tedious and cumbersome when multiple reactions are performed and also can lead to contamination of PCR reactions since tubes in close proximity to one another must be opened and closed manually by the operator in order to introduce the omitted component.
In another hot-start method, all of the necessary components are assembled in the reaction mixture at room temperature, but one critical component is physically isolated from the remainder of the reaction mixture using a barrier material that will melt or dissolve at elevated temperatures. Once the barrier material, typically a wax, has melted, the isolated component is introduced into the remainder of the reaction mixture and the primer extension reaction can proceed at the more stringent temperature. Conventionally, the polymerase is isolated using the barrier or wax material. This method, which is described in detail in U.S. Pat. No. 5,411,876 and Chou et al., Nucleic Acids Research 20(7):1717-1723 (1992), allows more specific amplification but is cumbersome in the set-up and implementation of the barrier material.
Another method is to use an antibody that non-covalently binds to the polymerase and prevents its activity at lower temperatures. At higher temperatures, the non-covalent bond between the antibody and the polymerase is disrupted and polymerase activity is restored for the rest of the PCR reaction. This method is further described in U.S. Pat. No. 5,338,671. Although this method is effective, the production process for generating the antibody is expensive and can introduce contaminating mammalian genomic DNA into the PCR reaction.
Yet another technique involves covalent attachment of a chemical moiety to the polymerase which blocks its activity at lower temperatures. This covalent bond can be broken after significant heating (e.g., above 95° C. for about 10-15 minutes) after which the polymerase activity is restored. A variety of chemical modifications can be introduced to produce the polymerase-moiety complex required to practice this technique as described in U.S. Pat. Nos. 5,677,152, 6,183,998 and 6,479,264. This technique has the disadvantage of requiring an extensive initial heating step which can damage DNA through heat-induced depurination. Such an extensive heating step also markedly reduces the activity of the polymerase relative to standard PCR methods.
In summary, primer extension reactions can be defined by two key events. One, the process of hybridizing the primer to the template and two, the extension of the hybrid by the catalytic action of a polymerase. The specificity of the hybridization is governed by the principles of thermodynamics in which lower temperatures favor nonspecific priming and amplification artifacts. Because polymerase chain reactions are conventionally assembled at lower temperatures, amplification artifacts can be a problem. Various methods have been developed to address this problem, techniques known as hot-start PCR. The present invention is a novel method of hot-start PCR.